Clostridium difficile (C. difficile) is a common nosocomial pathogen and a major cause of morbidity and mortality among hospitalized patients throughout the world. Kelly et al., New Eng. J. Med., 330:257-62, 1994. The increased use of broad spectrum antibiotics and the emergence of unusually virulent strains of C. difficile have lead to the idea that immunotherapies may be well suited to reduce disease and death associated with this bacterium. C. difficile has few traditional antibiotic options and frequently causes a recurring disease (25% of cases). Even with medical intervention, C. difficile claims about 20,000 lives in the USA alone per year and causes around 500,000 confirmed infections. Recently, more virulent strains of C. difficile have emerged that produce elevated levels of toxin such as the B1/NAP1/027 strain, which also has a decreased susceptibility to metronidazole. Outbreaks of C. difficile have necessitated ward and partial hospital closure due to the persistence of the spores that facilitate the spread of the disease. With the increasing elderly population and the changing demographics of the population, C. difficile is set to become a major problem in the 21st century. The spectrum of C. difficile disease ranges from asymptomatic carriage to mild diarrhea to fulminant pseudomembranous colitis.
C. difficile has a dimorphic lifecycle whereby it exists both as an infectious and tough spore form and a metabolically active toxin-producing vegetative cell. C. difficile-associated disease (CDAD) is believed to be caused by the vegetative cells and more specifically the actions of two toxins, enterotoxin toxin A and cytotoxin toxin B. To date, vaccines and immune therapy for C. difficile have focused upon the toxins (A and B), toxoids of A and B, recombinant fragments of A and B, and vegetative cell surface layer proteins (SLPAs).
Toxin B (TcdB, ˜269 kDa) is an approximately 2366 residue single polypeptide toxin encoded on a C. difficile pathogenicity locus (PaLoc) that also includes genes for two regulators (TcdC and TcdR) of toxin expression, a putative holin (TcdE), and Toxin A (TcdA). TcdB has at least four functional domains that contribute to cell entry and glucosylation of small-GTPases within the cytosol of the cell. TcdB's glucosyltransferase domain is included in the first 543 residues of the toxin and is the biologically active domain, which also includes a conserved DXD motif (Asp286/Asp288) and Trp102, which form a complex with Mn2+ and UDP-Glucose. The cysteine protease domain at residues 544-955 is necessary for autoproteolytic activity and delivery of the enzymatic domain into the cytosol. Between the cysteine protease domain and the C-terminal binding domain, a delivery domain is suggested, which is responsible for toxin translocation across membranes and delivers the glucosyltransferase into the cytosol of target cells. Finally, the fourth functional domain of TcdB is located within the carboxy-terminal region of the toxin (1851-2366), and is predicted to interact with receptors on target cells. However, the precise toxin receptor in humans has not been identified. After binding, the toxins are endocytosed via clathrin- and dynamin-dependent pathways to reach acidic endosomal compartments from where the toxins are translocated into the cytosol. Most likely in the cytosol, the cysteine protease domain is activated by binding of InsP6, resulting in autocleavage and release of the glucosyltransferase domain, which then targets Rho proteins (RhoA, -B, and -C), Rac and Cdc42. The glycosylation of these small regulatory proteins, lead to disruption of vital signaling pathways in the cells, resulting in actin condensation and consequent rounding of the cells, membrane blebbing, and eventual apoptosis and death of the target cell. While both TcdA and TcdB exert their activities on a wide range of cell types, TcdB exhibits a higher rate of enzymatic activity than TcdA, leading to a quickened rate of cytopathic effects in some cell types. Depending on the cell type, TcdB ranged from 4-fold to 200-fold more cytotoxic than TcdA in different studies.
There is an unmet need for effective treatment and/or prevention of C. difficile associated infections including prevention from relapse of CDAD. The present invention provides both mouse and humanized antibodies to toxin B to satisfy these and other needs.